Biology subjects

Connolly, J. P.

Publications and source records attributed to Connolly, J. P..

7 recordsLinked to original sources

bioRxiv2026

A diverse family of protein antibiotics inhibits the BAM complex by β-signal mimicry

Lectin-like protein antibiotics (Llps) kill Pseudomonas by inhibiting BamA, the essential core of the beta-barrel assembly machinery (BAM). Llps are composed of one or two beta;-lectin domains and a C-terminal peptide, with characterisation to date centred on the two-domain L-type pyocins of P. aeruginosa, which bind BamA extracellular loop 6 and inhibit by delivering the C-terminal peptide to BamA beta;-strand 1. Whether this mechanism is general, and how Llps contend with BamA sequence differences across species, remained unknown. We screened 238 Pseudomonas Llps against 101 genome-sequenced isolates, showing susceptibility across the family is set by BamA extracellular loop 6. Loop 6 length varies, and Llps fall into three targeting modes: short-loop and long-loop specialists and broad-range dual targeters, which have arisen repeatedly. We determine the 2.66 angstrom cryo-electron microscopy structure of a two-domain LlpA bound to BAM, showing recognition has been relocated relative to the L-type pyocins, with loop 6 bound by a single face of the N-terminal beta-lectin domain rather than the inter-domain cleft. This explains how single-domain LlpBs retain BamA targeting and why the second lectin domain has been lost at least twice. We show that the BamA inhibition mechanism is shared across Llps, with a hypervariable C-terminal peptide binding BamA beta-strand 1, inhibiting the BAM insertase by mimicking the beta-signal of BamA substrates. Together, these data provide an atlas of Llp diversity and the mechanistic principles for engineering it, identifying Llps as a tuneable, multi-interface scaffold for BAM-directed precision antibacterials against pathogenic Pseudomonas, including economically important plant pathogens.

biochemistry↗

bioRxiv2025

A Protein Antibiotic Inhibits the BAM Complex to Kill Without Cell Entry

Many antibiotics are ineffective against Gram-negative pathogens such as Pseudomonas aeruginosa because they cannot penetrate the bacterial outer membrane. Here, we show that protein antibiotics called L-type pyocins kill P. aeruginosa by inhibiting the {beta}-barrel assembly machinery (BAM) complex at the cell surface, halting outer-membrane protein assembly. Using single-particle cryo-electron microscopy, we show that L-type pyocins bind a surface-exposed region of BamA and deploy a C-terminal peptide that competitively inhibits the BAM complex, demonstrating that cell entry is not required for antibiotic activity. We combine genetics, multi-omics and cryo-electron tomography to show that BAM complex inhibition by L-type pyocins or the peptide antibiotic darobactin triggers a multifaceted transcriptomic, proteomic and morphological response. Despite this, BAM inhibition ultimately leads to a catastrophic loss of membrane integrity and cell death. These results validate BAM as a target for antibiotics that do not enter the cell and define an engineerable system for their development.

microbiology↗

bioRxiv2025

Convergent evolution of distinct D-ribulose utilisation pathways in attaching and effacing pathogens

Attaching and effacing pathogens overcome colonisation resistance by competing with metabolically similar organisms for limited resources. Enterohaemorrhagic E. coli (EHEC) utilises the pathogenicity island-encoded Accessory -arabinose Uptake (Aau) transporter to effectively colonise the mouse gut, hypothesised to be achieved via an enhanced capacity to scavenge -arabinose. Aau is regulated exclusively in response to -arabinose, but it is unclear how this system specifically benefits EHEC in vivo. Here, we show that Aau displays a >200-fold higher affinity for the monosaccharide D-ribulose, over -arabinose. EHEC cannot grow on D-ribulose as a sole carbon source and this sugar does not trigger aau transcription. However, Aau effectively transports D-ribulose into the cell only in the presence of -arabinose, where it feeds into the pentose phosphate pathway, after phosphorylation by the -ribulokinase AraB, thus providing EHEC a significant fitness advantage. EHEC has therefore evolved a mechanism of hijacking the canonical -arabinose utilisation machinery to promote D-ribulose utilisation in vivo. Furthermore, Citrobacter rodentium encodes an analogous system that exclusively transports D-ribulose and metabolises it via a dedicated D-ribulokinase. These unique mechanisms of D-ribulose utilisation suggest that convergent evolution has driven the ability of distinct pathogenic species to exploit this nutrient during invasion of the gut niche.

microbiology↗

bioRxiv2023

Metabolism of ʟ -arabinose converges with virulence regulation to promote enteric pathogen fitness

Virulence and metabolism are often interlinked to control the expression of essential colonisation factors in response to host-associated signals. Here, we identified a novel transporter of the dietary monosaccharide -arabinose that is widely encoded by the zoonotic pathogen enterohaemorrhagic Escherichia coli (EHEC), required for full competitive fitness in the mouse gut and highly expressed during human infection. Accordingly, we discovered that -arabinose induces expression of the EHEC type 3 secretion system, enhancing its ability to attach to host cells, and that the underlying mechanism is dependent on products of its catabolism rather than the sensing of -arabinose as a signal. Finally, using the murine pathogen Citrobacter rodentium, we show that -arabinose metabolism provides a fitness benefit during enteric infection via virulence factor regulation, as opposed to supporting pathogen growth. This study describes an intrinsic mechanism of integrating central sugar metabolism with virulence regulation and highlights the unexpected impact that nutrient utilisation can have in enteric pathogens.

microbiology↗

bioRxiv2021

D-serine induces distinct transcriptomes in diverse Escherichia coli pathotypes

Appropriate interpretation of environmental signals facilitates niche specificity in pathogenic bacteria. However, the responses of niche-specific pathogens to common host signals are poorly understood. D-serine (D-ser) is a toxic metabolite present in highly variable concentrations at different colonisation sites within the human host that we previously found is capable of inducing changes in gene expression. In this study, we made the striking observation that the global transcriptional response of three Escherichia coli pathotypes - enterohaemorrhagic E. coli (EHEC), uropathogenic E. coli (UPEC) and neonatal meningitis associated E. coli (NMEC) - to D-ser was highly distinct. In fact, we identified no single differentially expressed gene common to all three strains. We observed the induction of ribosome-associated genes in extraintestinal pathogens UPEC and NMEC only, and the induction of purine metabolism genes in gut-restricted EHEC and UPEC indicating distinct transcriptional responses to a common signal. UPEC and NMEC encode dsdCXA - a genetic locus required for the detoxification and hence normal growth in the presence of D-ser. Specific transcriptional responses were induced in strains accumulating D-ser (WT EHEC and UPEC/NMEC mutants lacking the D-ser-responsive transcriptional activator DsdC), corroborating the notion that D-ser is an unfavourable metabolite if not metabolized. Importantly, many of the UPEC-associated transcriptome alterations correlate with published data on the urinary transcriptome, supporting the hypothesis that D-ser sensing forms a key part of urinary niche adaptation in this pathotype. Collectively, our results demonstrate distinct pleiotropic responses to a common metabolite in diverse E. coli pathotypes, with important implications for niche selectivity. ImportanceThe pathogenic Escherichia coli comprise a group of highly specialized bacteria, some of which are capable of disseminating from the intestine and causing disease at other sites within the human host. Chemicals (metabolites) derived from the host and other microorganisms shape the behaviour of E. coli in different environments. Here we investigate the changes in gene expression that occur in E. coli strains capable (UPEC and NMEC) and incapable (EHEC) of metabolizing D-ser - a metabolite specifically enriched in the urine and in regions of the brain. We show that EHEC, UPEC and NMEC - distinct pathotypes associated with disease in the gut, bladder and brain, respectively, respond in a distinct manner to D-ser. Many of the genes affected by D-ser have been shown to be important during disease, highlighting the importance of varied responses to this common signal in host infection.

molecular biology↗

bioRxiv2018

A highly conserved complete accessory Escherichia coli type III secretion system 2 is widespread in bloodstream isolates of the ST69 lineage

Bacterial type III secretion systems (T3SS) play an important role in pathogenesis of Gram-negative infections. Enteropathogenic and enterohemorrhagic Escherichia coli contain a well-defined T3SS but in addition a second T3SS termed E. coli T3SS 2 (ETT2) has been described in a number of strains of E. coli. The majority of E. coli contain elements of a genetic locus encoding ETT2, but which has undergone significant mutational attrition rendering it without predicted function. Only a very few strains have been reported to contain an intact ETT2 locus. To investigate the occurrence of the ETT2 locus in strains of human pathogenic E. coli, we carried out genomic sequencing of 162 isolates obtained from patient blood cultures in Scotland. We found that all 26 ST69 isolates from this collection contained an intact ETT2 together with an associated eip locus which encodes putative secreted ETT2 effectors as well as eilA, a gene encoding a putative transcriptional regulator of ETT2 associated genes. Using a reporter gene for eilA activation, we defined conditions under which this gene was differentially activated. However, comparison of secreted proteins from ST69 strains under high and low eilA activation failed to identify any ETT2 secreted substrates. The conservation of the genes encoding ETT2 in human pathogenic ST69 strains strongly suggests it has functional importance in infection, although its exact functional role remains obscure.\n\nImportanceOne of the commonest bacteria causing bloodstream infections in humans is Escherichia coli, which has a significant morbidity and mortality. Better understating of the mechanisms by which this microbe can invade blood could lead to more effective prevention and treatment. One mechanism by which some strains cause disease is by elaboration of a specialized secretion system, the type III secretion system (T3SS), encoded by the locus of enterocyte effacement (LEE). In addition to this well-defined T3SS, a second T3SS has been found in some E. coli strains termed E. coli type III secretion system 2 (ETT2). Most strains carry elements of the ETT2 locus, but with significant mutational attrition rendering it functionless. The significance of our work is that we have discovered that human bloodstream isolates of E. coli of sequence type 69 contain a fully intact ETT2 and associated genes, strongly suggesting its functional importance in human infection.

microbiology↗

bioRxiv2018

Propionic acid enhances the virulence of Crohn’s disease-associated adherent-invasive Escherichia coli

The short chain fatty acid propionic acid (PA) is a bacteria-derived human intestinal antimicrobial and immune modulator used widely in Western food production and agriculture. Here we examine the effect of PA on the pathogenicity of the Crohns disease-associated microbe, adherent-invasive Escherichia coli (AIEC). Passage of AIEC through a murine model, where the low intestinal PA levels were increased to replicate those of the human intestine, led to the recovery of AIEC post-infection that had significantly increased virulence. These phenotypic changes, including increased adhesion to intestinal epithelial cells and biofilm formation, could be replicated in AIEC in vitro through exposure to PA alone. This in vitro exposure of AIEC to PA fundamentally changed AIEC virulence, with strains exposed to PA in vitro subsequently persisting at 20-fold higher levels in a murine model compared to non-exposed strains. RNA-sequencing identified the transcriptional changes in AIEC in response to PA with upregulation of genes involved in biofilm formation, stress responses, metabolism, membrane integrity and alternative carbon source utilisation. These PA induced changes in virulence could be replicated in a number of E. coli isolates from Crohns disease patients. Finally, removal of the PA selective pressure was sufficient to reverse these phenotypic changes. Our data indicate that exposure of AIEC to PA evolves bacteria that are both resistant to this natural human intestinal antimicrobial and increasingly virulent in its presence.\n\nImportanceExposure to propionic acid, an intestinal short chain fatty acid and commonly used antimicrobial in Western food production, induces significant virulence associated phenotypic changes in adherent-invasive Escherichia coli (AIEC).

microbiology↗